ON THE USE OF CHITOSAN-IMMOBILIZED BETA-GLUCOSIDASE IN WINE-MAKING - KINETICS AND ENZYME-INHIBITION

The kinetics of chitosan-immobilized beta-glucosidase and enzyme inhib ition by several components of wine and must (glucose, fructose and te rpenols) were studied. Optimum immobilization conditions were: tempera ture 25 degrees C, pH between 5.5 and 6.0, polymeric support dimension in the range 38-75 mu m, cross-linking time 30 min, glutaraldehyde co ncentration 0.5-1.0% w/v, 1 g of chitosan per 1000 units of beta-gluco sidase. The immobilized enzyme retained 29% of the wet biocatalyst act ivity when freeze-dried and showed good stability (half-life roughly 2 years) when stored at 4 degrees C. Kinetics were tested at 25 degrees C following the hydrolysis of beta-nitrophenyl beta-D glucopyranoside and obey the Michaelis-Menten rate equation. K-m = 1.3 mM and the act ivation energy, 62.84 kJ mol(-1), are close to those of the free enzym e. The operational half-life was roughly 500 h. Glucose only depressed the enzyme activity according to a reversible non-competitive inhibit ion mechanism with K-i = 11.2 mM.